人肠三叶因子在pET系统中的表达、纯化及生物活性  

Expression,purification and biological activity of human intestinal trefoil factor in pET system

作　　者：孙勇[1,2] 王良喜[1,2] 孙曙光[1,2] 毛学飞[1,2] 邓向东[1,2] 潘晓峰[1,2] 张方[1,2] 

机构地区：[1]徐州医学院附属淮海医院 [2]中国人民解放军第97医院烧伤整形科,江苏徐州221004

出　　处：《山东大学学报（医学版）》2014年第1期23-28,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81100252);南京军区医学科研课题重点项目(09Z007;12Z10);中国人民解放军第九七医院院管课题

摘　　要：目的构建人肠三叶因子(hITF)原核表达载体,表达重组hITF,并考察其生物学活性。方法通过RT-PCR获得hITF cDNA片段,将目的基因插入原核表达载体pET32a,得到重组载体pET32a-hITF,优化表达条件获得最大表达产量,Ni-NTA亲和层析一步法纯化重组蛋白,SDS-PAGE、Western blotting及N端测序鉴定重组蛋白,细胞重建模型验证其功能。结果经PCR及测序证实,hITF cDNA准确插入原核表达载体pET32a中,诱导表达后,SDS-PAGE分析证明hITF的分子量约为32kD,Western blotting分析表明,表达蛋白具有良好的抗原性和特异性;N端15个氨基酸测序证明其序列正确性;体外实验证实其具有细胞促迁移能力。结论成功构建出原核表达载体pET32a-hITF,获得重组hITF。Objective To construct Escherichia coli （E. coli） expression vector of human intestinal trefoil factor （hITF）, express recombinant hITF and analyze its biological activity. Methods The hITF gene encoding mature pep- tide was amplified by RT-PCR, and then inserted into the expression vector pET32a. Recombinant plasmid pET32a- hITF was transformed into the Escherichia coli Origami B （ DE3 ） and hITF was expressed by IPTG induction, hITF was purified by Ni-NTA affinity chromatography, and determined by SDS-PAGE, Western blotting, and N-terminal amino acid sequence analysis. Biological activity was assayed in an in vitro restitution model. Results It was proved that the fragment amplified was inserted into the expression vector pET32a correctly by PCR and gene sequencing. SDS-PAGE analysis proved that the molecular weight of hITF was about 32 kD, and Western blotting demonstrated that the expres- sive proteins had good antigenicity and specificity. The N-terminal 15 amino acid sequence was consistent with the theoretical value. In addition, hITF was proved to enhance migration activity. Conclusion hITF Escherichia coil expression vector is successfully constructed and recombinant hITF is expressed.

关 键 词：人肠三叶因子 大肠杆菌 诱导表达 

分 类 号：Q786[生物学—分子生物学]